Compromised vascular perfusion is a major factor associated with progression of many disease states, failure of tissue/organ transplants, and complications related to wound repair. At present, cell-based vasculogenesis strategies involving endothelial precursors continue to show promise but suffer from suboptimal delivery strategies that promote localization, survival, and predictable guidance of vessel formation by therapeutic cells. Furthermore, definition of essential therapeutic cell attributes is lacking and likely contributes to observed variability in preclinical and clinical outcomes. The long-term goal of the proposed work is to develop a collagen- based, cell-delivery matrix that predictably induces three-dimensional (3D) vessel formation through tunable biophysical, vascular-inductive features. The proposed work uniquely interfaces an innovative collagen polymer engineering approach that focuses on naturally-occurring collagen intermolecular cross-links and endothelial colony forming cells (ECFC), a specific population of endothelial precursors defined by their high proliferative and vessel forming capacities. The proposal objective is to define how cross-links modulate collagen assembly and define specific matrix biophysical features that can be tuned for guiding ECFC vessel formation in vitro and in vivo. The proposed activities involve three aims 1) Define how the intermolecular cross-link composition of collagen polymers modulates the molecular assembly of collagen-fibril matrices and contributes to tunability of fibril- and matrix-level biophysical features known to be important to vessel morphogenesis;2) Define how fibril- and matrix-level design features including, fibril density, interfibril branching, stiffness, and biodegradability work independently and interdependently to modulate early- and late-stage processes of ECFC vessel formation in vitro and in vivo;and 3) Define critical nodes within the matrix-integrin-cytoskeleton signaling axis, including 21-integrin, FAK, Cdc42, and MT1-MMP and their roles as molecular mechanisms by which ECFC sense and respond to matrix biophysical cues and potential pathways to modulate vessel formation. Purified collagen polymers, specified in terms of their intermolecular cross-link composition, will be isolated and prepared from pig skin and tendon. Both collagen concentration and cross-link composition will be systematically varied to define how these polymerization parameters alter assembly kinetics and biophysical properties of resultant matrices. These collagens will then be used to suspend ECFC to define how specific matrix biophysical features affect vessel formation and persistence in vitro and in vivo. Finally, experiments involving outside-in and inside-out perturbation strategies will be conducted to identify critical nodes of signaling mechanisms by which ECFC sense, prioritize, and respond to matrix biophysical cues. Collectively, the knowledge and perspective gained is expected to significantly impact tissue engineering and regenerative medicine by refining how collagens are characterized, standardized, and applied to the rationale design of vascular-inductive matrices. PUBLIC HEALTH RELEVANCE: Our innovative and multidisciplinary approach will provide the first in-depth study documenting how intermolecular cross-links constitute a valid collagen polymer design parameter for tuning matrix biophysical properties known to be important to vessel morphogenesis. The interface of a novel set of collagen building blocks with the high proliferative potential and vessel forming capacity of endothelial colony forming cells (ECFC) is expected to positively impact the fields of tissue engineering and regenerative medicine by providing 1) novel collagen-based, delivery matrices designed to polymerize in situ so to localize human ECFC and provide them with customizable biophysical vascular-inductive cues and 2) vascularized tissue modules to support development and production of clinical-scale engineered tissue/organ replacements.